Pathologic angiogenesis plays a pivotal role in the progression of a variety of diseases. Our previous work has demonstrated that neonatal hypoxic pulmonary arterial hypertension is a condition involving pulmonary artery adventitial thickening, accumulation of inflammatory blood cells, and neovascularization of the adventitial vasa vasorum [3
]. Although many factors with angiogenic potential have been described, factors specifically involved in hypoxia-induced expansion of the vasa vasorum neovascularization in the PA have not been delineated. The findings of the present study provide strong support for the idea that extracellular ATP, potentially released from a variety of cells in the adventitia, including the vasa vasorum endothelial cells themselves, may be involved in this process. Our findings consistently showed that extracellular ATP robustly stimulated angiogenic responses, i.e., proliferation, migration, and network formation in Matrigel in VVEC derived from at least six different chronically hypoxic animals with pulmonary hypertension, adventitial remodeling, and vasa vasorum expansion. Interestingly, ATP had little angiogenic effects on main pulmonary artery endothelial cells or aortic endothelial cells isolated from the same animals. Importantly in VVEC, extracellular ATP potentiates the effect of VEGF and bFGF, other angiogenic factors known to be produced in hypoxic microenvironment. We found that ATP activated ERK1/2, PI3K/Akt, and mTOR/p70S6K to a greater degree in VVEC than in AOEC and MPAEC, and that these signaling pathways are critical in ATP-induced proliferative and migratory responses in VVEC. Collectively, our data support the idea that extracellular ATP participates in the expansion of the vasa vasorum that is observed in the pulmonary artery adventitia of chronically hypoxic neonatal calves.
Although extracellular nucleotide-mediated signaling has been implicated in various physiological and pathologic conditions, there is limited information regarding the role of extracellular nucleotides in modulating endothelial cell proliferation. We found that extracellular ATP stimulates dramatic increases in DNA synthesis in VVEC, but has very little effect on AOEC and MPAEC isolated from the same animals. The exceptional sensitivity of microvascular endothelial cells to stimulation with ATP is supported by other studies showing a mitogenic effect of extracellular ATP, UTP, and 2MeSATP on brain capillary and corneal endothelial cells [45
]. The exaggerated responses to extracellular ATP suggest that VVEC may be abundantly equipped with multiple
purinergic receptors. However, unexpectedly neither UTP nor UDP stimulated DNA synthesis in VVEC, though a previous report demonstrated both these pyrimidines stimulate guinea pig cardiac endothelial cell proliferation [23
]. Analysis of nucleotide efficacy revealed that the P2Y1 family of purinergic receptors, P2Y1, P2Y11, P2Y12, and P2Y13 and perhaps, P1 adenosine receptors, but not pyrimidine P2Y4 and P2Y6 receptors, play a predominant role in VVEC mitogenesis. Thus, VVEC appears to display a distinct phenotype characterized by the expression of a specific subset of purinergic receptors. Interestingly, expression of the subsets of P2Y1-like purinergic receptors has been reported for several types of immune and circulating blood cells [24
]. When considered in the context of our previous findings demonstrating the presence of c-kit + cells in the newly expanding vasa vasorum [3
], these data support the idea that vasa vasorum neovascularization might involve both angiogenesis and vasculogenic processes and that extracellular ATP could be involved in both.
Previous studies have demonstrated that endothelial cells derived from large vessels express several types of purinergic receptors. Expression of P2Y1 and P2Y2 receptors has been shown in bovine aortic endothelial cells and expression of P2Y1, P2Y2, P2Y6, P2X4, and P2X5 receptors documented in human umbilical vein endothelial cells [47
]. Activation of P2 receptors in these cells mediates the release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), prostacyclin, and t-PA [47
]. Similar to our observations in AOEC and MPAEC, Daele et al. have found that ATP and synthetic P2Y receptor agonists exerted only weak increases in DNA synthesis in bovine aortic endothelial cells [49
]. In addition to adenine nucleotides, pyrimidine nucleotides, UTP and UDP have been shown to exert mitogenic and chemotactic effects on human umbilical vein and guinee pig cardiac endothelial cells [20
]. However, in our studies we did not observe any stimulatory effect of UTP and UDP, on DNA synthesis in any of the examined endothelial cell types. Thus our data, when combined with that from other reports, suggest that there must be tremendous heterogeneity in endothelial phenotypes, with regard to extracellular nucleotide-induced functional responses and the signaling pathways through which these effects are mediated.
To further investigate the possibility that differences in post-receptor signaling events in VVEC, AOEC and MPAEC might be involved in conferring distinct proliferative and migratory phenotypes, we evaluated the activation of intracellular kinases downstream of P2Y receptors. We found significant differences in the magnitude and duration of activation of ERK1/2, Akt, mTOR, and p70S6K between VVEC and AOEC and MPAEC. Greater and more prolonged activation of EKR1/2 and p70S6K in VVEC could contribute to proliferative and migratory advantages. In fact, we demonstrated that blockade of these pathways inhibited proliferation and migration. Interestingly, we also observed that ATP in combination with VEGF or bFGF resulted in more dramatic induction of DNA synthesis in VVEC than in other endothelial cell types. This observation suggests that under conditions of chronic hypoxia, the simultaneous release of mitogenic growth factors and nucleotides from vascular and circulating blood cells may result in cumulative stimulatory effects on proliferation and migration of specific endothelial cells, in our case the VVEC.
Enzymatic reactions contributing to the pathways of extracellular ATP synthesis and degradation play a critical role in regulating the concentration of extracellular ATP and its metabolites near purinergic receptors. Recent studies have implicated the endothelial cell surface ecto-F1
ATP synthase in endothelial cell and tumor cell proliferation [50
]. It has been shown that both angiostatin and monoclonal antibodies directed against the β-subunit of ecto-F1
ATP synthase-inhibited human umbilical vein endothelial cell proliferation through inactivation of extracellular ATP synthetic pathway [52
]. Intringingly, endothelial progenitor cells were found to be more sensitive to angiostatin than mature endothelial cells [53
]. Given the observations that ATP seems to exert such potent effects on VVEC proliferation as well as the possibility that circulating endothelial precursors might be incorporated at the sites of neovascularization [3
], future experiments directed at an evaluation of ATP synthesis and degradation pathways on VVEC surface will be important.
Morphogenetic changes in endothelial cells are critical for appropriate assembly in newly forming blood vessels. It is believed that formation of a tube-like intercellular network on basement membrane matrix (Matrigel) reflects the ability of endothelial cells to undergo differentiation. To date, there have been no studies to demonstrate the effect of extracellular ATP on endothelial rearrengement into the tube-like networks. Using in vitro Matrigel assay on growth factor-reduced matrix, we demonstrated that extracellular ATP exerted significant effects on VVEC tube-like network formation. Interestingly, under basal conditions, AOEC and MPAEC rearrange in tube-like networks with a patterns different than those observed for VVEC. However, in contrast to VVEC, ATP did not exert significant effects on tube-like formation in these cells. The reasons for these different responses in Matrigel under both basal and ATP-stimulated conditions exhibited by the different cell types remain unclear and will require future investigation. They do, however, reinforce the idea that endothelial cells isolated from different vascular beds, maintain distinct functional characteristics even after being perpetuated in culture. The maintenance of these differences will allow evaluation of vascular bed-specific endothelial phenotypes in vitro [54
Characterization of the signaling pathways contributing to angiogenesis is important in developing pharmacologic strategies for both pro- and anti-angiogenic therapy. It has been shown that constitutive activation of PI3K/Akt/mTOR and ERK pathways is observed under various pathological conditions associated with chronic hypoxia, inflammation, and cancer, suggesting that in the hypoxic adventitial microenvironment, these pathways could play a permissive role for vasa vasorum growth [43
]. It has been previously reported by others and us that ERK1/2 and PI3K pathways play a critical role in extracellular ATP-induced proliferation of smooth muscle and fibroblasts [31
]. However, the involvement of PI3K and mTOR pathways in nucleotide-mediated angiogenic responses in endothelial cells has not been fully investigated. The results of our studies demonstrate that extracellular ATP activates PI3K/Akt, mTOR/p70S6K, and ERK1/2 pathways in VVEC and that activation of these pathways contributes to VVEC mitogenic and migratory responses. Using PI3K isoform-specific antibodies and in vitro kinase assay, we found that ATP-stimulated PI3K activity is selectively associated with the p85 regulatory subunit and p110β catalytic subunit, indicating that at least class I PI3Kβ is involved in ATP-induced mitogenic signaling events in VVEC. Our observations also suggest a possibility that PI3Kδ, an isoform expressed predominantly in hematopoietic cells, may play a role in mediating the effects of extracellular ATP and/or another angiogenic factors in VVEC. Complementary to our findings on the role of PI3K pathway in endothelial angiogenic responses, studies on human umbilical vein endothelial cells (HUVEC) demonstrated the involvement of PI3K, focal adhesion kinase, p130cas
, and paxillin in extracellular ATP- and UTP-mediated cell migration [20
]. In addition, activation of ERK and PI3K/PDK/PKC pathways in response to P2Y receptor agonists was demonstrated in HUVEC and in U138-MG human glyoma cell line, demonstrating the ubiquitous role of extracellular nucleotides and associated PI3K-dependent pathways in regulating growth responses. Finally, our data demonstrate that ERK1/2, but not PI3K and mTOR, contributes to extracellular ATP-induced VVEC rearrangement into tube-like networks in Matrigel, suggesting that additional, as yet unidentified signaling pathways are involved in endothelial tubulogenesis. Recent observations in aortic and lung microvascular endothelial cells demonstrate that PLCγ/Ca2+
and cAMP/ERK1/2 pathways may be considered for their role in the endothelial tube-like formation [59
]. Because ATP-mediated activation of VVEC involves increases in intracellular Ca2+
level (unpublished observation), evaluation of PLC/Ca2+
pathway will be an important focus of future studies.
In conclusion, our findings also demonstrate that extracellular ATP is potent angiogenic factor for VVEC, but not for endothelial cells derived from large systemic and pulmonary vessels, such as aorta and main pulmonary artery. The dramatic effect of extracellular ATP on the activation of PI3K/Akt/mTOR and ERK1/2 pathways suggests that in VVEC, expression of the components of these signaling pathways together with P2Y1 purinergic receptors, contributes to a pro-angiogenic phenotype. Finally, the results of our studies indicate potential significance for anti-angiogenic therapy, in which a combination of purinergic receptor antagonists with PI3K/mTOR- and ERK1/2-specific inhibitors may be considered for eliminating neovessel growth under pathological conditions where elevated levels of extracellular ATP and the other purine nucleotides can be expected.